Bax bs 0127r

To get cells lysates containing the total proteins, the AGS cells were lysed with RIPA lysis solution. The concentration of the proteins was quantified using the Bradford assay. The samples were applied to 10% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), transferred to a PVDE (poly vinylidene difluoride) membrane. The membrane was then immersed in TBST (Tris-buffered saline and Tween) containing 5% defatted milk for 1 h with gentle agitation and incubated overnight at 4 °C with the primary antibody (GAPDH #60004-Ig purchased from Proteintech; AKT #YT0185; p-AKT #YP0006; mTOR #YT2913 purchased from ImmunoWay; PI3K# YP0176 purchased from Affinity; Bcl-2 #bs-0032R; Bax #bs-0127R; Caspase-3# bs-0081R purchased from Bioss). After that, the membrane was incubated with HRP-labeled (horseradish peroxidase) secondary antibodies for 2 h at room temperature and detected on photographic film. Finally, the bands of the proteins were analysed with ImageJ software.

The paraffin-embedded sections of the neoplastic tissue were dewaxed, rehydrated, and antigen-repaired with sodium citrate for 30 min. Then, they were incubated with 3% hydrogen peroxide at room temperature for 20 min. Next, the sections were blocked with 3% BSA for 1 h and labeled with a primary antibody at 4 °C overnight. The following primary antibodies were used: CDK4 (11026-1-AP, Proteintech, Wuhan, China), CydlinD1 (26939-1-AP, Proteintech, Wuhan, China), CDC25A (55031-1-AP, Proteintech, Wuhan, China), Bax (bs-0127R, Bioss, Beijing, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), PCNA (bs-2006R, Bioss, Beijing, China), Ki67 (bs-23103R, Bioss, Beijing, China), B-Myb (ab12296, Abcam, Cambridge, United Kingdom), γ-H2A.X (bs-3185R. Bioss, Beijing, China), and p53 (bs-2090R, Bioss, Beijing, China). The sections were stained with iNOS, GBP5, and CD11b to analyze the phenotype of macrophages. The samples were subsequently stained with a secondary antibody (PV-9000, ZSGB-BIO, Beijing, China) at 37 °C for 1 h. Diaminobenzidine (DAB, ZL-9018, ZSGB-BIO, Beijing, China) was used for staining at room temperature for 1–3 min. The nuclei were stained with hematoxylin or DAPI. Finally, the paraffin-embedded sections were observed with an orthogonal Olympus microscope or a confocal microscope.